Molecular Markers of Pediatric Solid Tumors—Diagnosis, Optimizing Treatments, and Determining Susceptibility: Current State and Future Directions

Advances in molecular technologies, from genomics and transcriptomics to epigenetics, are providing unprecedented insight into the molecular landscape of pediatric tumors. Multi-omics approaches provide an opportunity to identify a wide spectrum of molecular alterations that account for the initiation of the neoplastic process in children, response to treatment and disease progression. The detection of molecular markers is crucial to assist clinicians in accurate tumor diagnosis, risk stratification, disease subtyping, prediction of treatment response, and surveillance, allowing also for personalized cancer management. This review summarizes the most recent developments in genomics research and their relevance to the field of pediatric oncology with the aim of generating an overview of the most important, from the clinical perspective, molecular markers for pediatric solid tumors. We present an overview of the molecular markers selected based on therapeutic protocols, guidelines from international committees and scientific societies, and published data.


Introduction
Recent decades have witnessed an intensive development of molecular research, which has contributed significantly to a more complete understanding of the molecular basis of childhood cancers. The published studies reveal a high heterogeneity of molecular alterations that account for the initiation of the neoplastic process, response to treatment and disease progression. These findings clearly indicate that the profile of molecular changes occurring in childhood malignancies differs significantly from the one observed in adult cancers. Thus, it is not possible to directly extrapolate the knowledge and experience with molecular markers from adults to the pediatric population. The differences observed pertain not only to the molecular basis, but also to the disease itself, its anatomical site and its histopathological features demanding the development of a different diagnostic and therapeutic approach for this group of patients.
Knowledge of specific childhood cancer genetic alterations present in tumor cells (somatic markers) as well as germline alterations is useful throughout the entire diagnostic and therapeutic process. There are genetic markers specific for histological types of cancer which are necessary for making a correct diagnosis (a group of diagnostic markers). Furthermore, there are molecular markers that correlate to the tumor's grade, allowing us to predict the course of disease and prognosis (a group of prognostic markers) as well as to predict responses to a specific treatment (a group of predictive markers). Most promising are the molecular markers for targeted therapies. Extensive research on the biology of cancers, including their molecular profile, has influenced the current way of practicing * of all malignant neoplasms of developmental age Since the mid-1970s, the cure rates for most pediatric solid tumors have increased by as much as 50% [4]. At present, more than 80% of children with cancer are cured. These excellent cure rates are achieved with multidrug chemotherapy combined with surgery and/or radiotherapy in the case of solid tumors. However, there is not much to offer to children with refractory or relapsed disease after first-or second-line treatment. For these patients, innovative and effective medicines are needed.
In recent decades personalized treatments based on molecular markers have been developed for adults with cancer. Nevertheless, not enough progress has been made in the development and authorization of targeted therapies for childhood malignancies. Though molecular markers are routinely used in diagnosis, establishing risk groups in pediatric oncology, and novel medicinal products are being developed, with some exceptions, few breakthrough medicines have come to the market for children. Only few such medicinal products received marketing authorization for the treatment of pediatric malignancies. Among them are ABL-class inhibitors for Philadelphia positive acute lymphoblastic leukemia, anti-CD antibodies for B cell lymphomas, and anti-GD2 antibodies for children with high-risk neuroblastoma. Recently, Larotrectinib for children with NTRK fusion solid tumors and Crizotinib for children and young adults with relapsed or refractory systemic anaplastic large-cell lymphoma received marketing authorization. Phase 2/3 studies with Crizotinib in children and adolescents with recurrent, progressive, and unresectable inflammatory myofibroblastic tumors are forthcoming.
Moreover, genetic data are lacking on the difficulty to treat refractory or relapsed solid tumors, limiting our knowledge of the molecular composition of such entities. To further improve cure rates in pediatric cancer it is essential to continue research and targeted medicine trials using tumor molecular profiling in children and adolescents.

Neuroblastoma
Neuroblastoma (NBL) is the most frequent pediatric extracranial tumor originating from neural crest progenitor cells. It accounts for approximately 10% of all childhood malignancies and for up to 15% of deaths in children from cancer [22]. It is the most common cancer diagnosed in children under 12 months of age with a median age at diagnosis of 17 months [23]. Neuroblastoma can present along the sympathetic nervous system, with the most common abdominal location in the adrenal gland or sympathetic chain. It is a heterogenous disease which is reflected in its clinical course of spontaneous regression, differentiation or rapid progression despite intensive multimodal treatment. Patients with low-and intermediate-risk neuroblastoma have good prognosis, with cure rates over 85%, while the survival of children with high-risk disease is less than 50%.
The most significant prognostic factors in NBL are the child's age at diagnosis, disease stage [4], tumor histology, DNA ploidy, MYCN amplification status and the presence of characteristic chromosomal aberrations (Table 2).
Recommendations are based on current therapeutic protocols and diagnostic guidelines from the European Neuroblastoma Group (SIOPEN group) and the INRG (International Neuroblastoma Risk Group Biology Committee) [24].
Recommendations are based on current therapeutic protocols and diagnostic guidelines from the European Neuroblastoma Group (SIOPEN group) and the INRG (International Neuroblastoma Risk Group Biology Committee) [24].
A set of molecular markers clinically relevant for neuroblastoma is provided in Table  2 [24][25][26][27][28]. The incidence of Wilms tumor is approximately 7 cases per 1 million children younger than 15 years of age, accounting for 5-7 percent of all childhood malignancies. WT can occur in both kidneys (bilateral disease), found in 5-8% of cases. The mean age at diagnosis is 44 months in unilateral cases and 31 months for bilateral cases of Wilms tumor. Wilms tumor is rare in patients older than age 15. A total of 1.5% of cases occur in related family members. Approximately 5% of WTs are associated with known constitutional predisposition syndromes.
Pathogenic changes in suppressors that regulate growth, differentiation and proliferation of embryonic kidney tissue play an essential role in the pathogenesis of this cancer. Alterations in WT1, CTNNB1 or AMER1 (WTX) genes are observed in about one-third of Wilms tumor cases [29,30]. Other important genes that regulate miRNA processing, such as DROSHA, DGCR8, DICER1 and XPO5, are also involved [31][32][33][34]. The third important group consists of certain genes, the expression of which plays a significant role in the early stages of kidney development, such as SIX1 i SIX2, EP300 (CREBBP), MLLT1, BCOR and MYCN. Alterations in the TRIM28 gene are associated with the epithelial subtype of Wilms tumor [35]. In anaplastic Wilms tumors, the presence of TP53 gene alterations is noted. An observed loss of heterozygosity within chromosome regions 1p and 16q as well as an increase in the amount of genetic material within chromosome 1q are associated with an unfavorable prognosis [36][37][38].
Recommendations are based on guidelines from two groups: Children Oncology Group (COG), continuing work of The National Wilms Tumor Study Group (NWTS) and The International Society of Paediatric Oncology-Renal Tumors Study Group (SIOP-RTSG), as well as the UMBRELLA therapeutic protocol and literature data [39][40][41][42].

Tumor Type Genes/Molecular Profiles Characteristically Altered
Diagnostic Marker

Malignant Bone Tumors, including Osteosarcoma and Ewing Sarcoma
Malignant bone tumors account for 6% of all childhood malignancies. The estimated incidence rate is 0.8 per million. The most common are osteosarcoma (56%), followed by Ewing sarcoma (34%) and chondrosarcoma (10%). The diagnosis of primary bone tumors relies on clinicopathological and radiological correlation. The recommended molecular assays include among others assessment of the presence of translocations characteristic of Ewing sarcoma and chondrosarcoma, determination of the status of pathogenic variants in the H3F3A gene that are relevant for the diagnostics of giant cell tumor of soft tissue, as well as assessment of the presence of MDM2, PRIM1, and CDK4 amplifications to differentiate lower grade osteosarcoma. The standard was prepared based on the current literature data [48][49][50][51].
A set of molecular markers clinically relevant for malignant bone tumors of childhood is listed in Table 4 [4,50-59].

Malignant Bone Tumors, including Osteosarcoma and Ewing Sarcoma
Malignant bone tumors account for 6% of all childhood malignancies. The estimated incidence rate is 0.8 per million. The most common are osteosarcoma (56%), followed by Ewing sarcoma (34%) and chondrosarcoma (10%). The diagnosis of primary bone tumors relies on clinicopathological and radiological correlation. The recommended molecular assays include among others assessment of the presence of translocations characteristic of Ewing sarcoma and chondrosarcoma, determination of the status of pathogenic variants in the H3F3A gene that are relevant for the diagnostics of giant cell tumor of soft tissue, as well as assessment of the presence of MDM2, PRIM1, and CDK4 amplifications to differentiate lower grade osteosarcoma. The standard was prepared based on the current literature data [48][49][50][51].

Soft-Tissue Sarcomas (STS)
Soft-tissue sarcomas are a heterogeneous group of malignant neoplasms that arise from embryonic mesenchymal and neuroectodermal tissue. They include neoplasms of muscle, connective and vascular tissue. Most cases occur in children aged 2-6 years and adolescents older than 12 years. The incidence rate, regardless of gender, ranges from 0.2-1.0/100,000 per year.
The most common STS in the pediatric group (70%) is rhabdomyosarcoma (RMS), with the age of onset usually before 10 years. The most common type of RMS is embryonal RMS. However, it is important to identify the alveolar RMS subtype for therapeutic management due to its worse prognosis. Recently, sclerosing and spindle cell rhabdomyosarcoma was separated as a stand-alone pathologic entity, in which two subtypes are molecularly defined: the infantile subset with VGLL2, TEAD1 and SRF fusion as well as the subset with p.L122R MYOD1 gene pathogenic variant. The presence of MYOD1 alteration is associated with poor outcomes and response to therapy [60].
The remaining neoplasms belong to the non-rhabdomyosarcoma soft tissue sarcoma NRSTS group, which is more common in older children and young adults. Synovial sarcoma and MPNST (malignant peripheral nerve sheath tumor) are the most common neoplasms in this group. Other pathological entities are much less prevalent in children.

Soft-Tissue Sarcomas (STS)
Soft-tissue sarcomas are a heterogeneous group of malignant neoplasms that arise from embryonic mesenchymal and neuroectodermal tissue. They include neoplasms of muscle, connective and vascular tissue. Most cases occur in children aged 2-6 years and adolescents older than 12 years. The incidence rate, regardless of gender, ranges from 0.2-1.0/100,000 per year.
The most common STS in the pediatric group (70%) is rhabdomyosarcoma (RMS), with the age of onset usually before 10 years. The most common type of RMS is embryonal RMS. However, it is important to identify the alveolar RMS subtype for therapeutic management due to its worse prognosis. Recently, sclerosing and spindle cell rhabdomyosarcoma was separated as a stand-alone pathologic entity, in which two subtypes are molecularly defined: the infantile subset with VGLL2, TEAD1 and SRF fusion as well as the subset with p.L122R MYOD1 gene pathogenic variant. The presence of MYOD1 alteration is associated with poor outcomes and response to therapy [60].
The remaining neoplasms belong to the non-rhabdomyosarcoma soft tissue sarcoma NRSTS group, which is more common in older children and young adults. Synovial sarcoma and MPNST (malignant peripheral nerve sheath tumor) are the most common neoplasms in this group. Other pathological entities are much less prevalent in children.
The recommendations were prepared based on the current literature data. A set of clinically relevant molecular markers for pediatric soft tissue sarcomas is provided in Table 5 [4,58,59,61].

Germ Cell Tumors
Germ cell tumors (GCTs) are derived from germ cells at different stages of their differentiation. They present a wide variety of site, histologic, and biological diversity. GCTs account for approximately 3-6% of all malignancies in children. The most common age of onset is between 1 and 6 years. Patients with GCT have good prognosis with cure rates over 85%.
Currently, the recommended molecular assays for germ cell tumors include karyotyping of patients with symptoms of gonadal dysgenesis, gonadoblastoma and mediastinal tumors (for boys) and bilateral ovarian tumors (for girls), in order to detect/rule out the presence of genetic syndromes such as Turner, Swyer, Klinefelter, Fraser, Denys-Drash and others. The literature data also indicate the presence of somatic rearrangements involving chromosomes 1, 6, 11, 12, 16, 20, and 22, the clinical significance of which is currently unknown.
The standards were developed following the guidelines of the French TGM-95 protocol (1995), protocols elaborated by the international expert consortium MaGIC-Malignant Germ Cell International Collaborative and literature data [62].

Liver Tumors
The most common pediatric liver tumor is hepatoblastoma. The incidence of hepatoblastoma is 2-3 cases per 1 million children under 15 years of age; however, approximately 98% of all cases occur in children under 5 years of age. The mean age at diagnosis is 18 months [63]. Survival rates for children with hepatoblastoma exceed 80%. Molecular alterations reported in hepatoblastoma primarily involve genes, the protein products of which regulate the WNT and NF-κB pathway. Thus, the most common somatic alterations are those in genes: CTNNB1 (80-90%), APC (2-3%), AXIN1, AXIN2 and PIK3CA as well as TERT (2-6%) and NFE2L2 (5-10%) [63][64][65][66][67][68][69]. The results of chromosomal rearrangement analysis indicate that this group of cancers has a higher rate of somatic rearrangements within chromosomes 1, 2, 8, and 20. However, the clinical significance of their occurrence is still unknown [64,65,70]. One of the objectives of the Pediatric Hepatic International Tumor Trial (PHITT (NCT03017326)), which has been running since 2017, is to determine the clinical significance of molecular findings including complete genomic, transcriptomic, and epigenomic profiling for hepatoblastoma patients [71].
Other hepatic tumors of childhood include: hepatocellular carcinoma (HCC) which is very rarely diagnosed in children, usually in older patients (10-14 years), but has been also found in children younger than 5 years of age. The prognosis is dismal, after conservative surgical treatment (30% of children achieving 3-year survival). Liver transplantation in children with HCC contributed to the improvement of overall survival (>70%). The most frequently reported somatic alterations in this cancer are alterations in the TERT and TP53 genes (60% and 25-30%, respectively); however, the molecular background is still unknown [72,73]; • undifferentiated embryonal sarcoma of the liver (UES) which is a rare liver tumor with onset in children mostly aged between 6 and 10 years. The molecular background of this neoplasm is not fully understood; however, according to literature data, the characteristic features of this neoplasm comprise frequent and extensive chromosome rearrangements, also in the form of chromothripsis [74]. Additionally, alterations were observed within the 19q13.4 region, including a t(11;19) (q13;q13.4) translocation and overexpression of the C19MC region (miRNA cluster). The presence of TP53 gene alterations was also observed [75][76][77][78]. The recommendations were prepared based on the current literature data. A set of clinically relevant molecular markers for pediatric soft tissue sarcomas is provided in Table 5 [4,58,59,61].

Germ Cell Tumors
Germ cell tumors (GCTs) are derived from germ cells at different stages of their differentiation. They present a wide variety of site, histologic, and biological diversity. GCTs account for approximately 3-6% of all malignancies in children. The most common age of onset is between 1 and 6 years. Patients with GCT have good prognosis with cure rates over 85%.
Currently, the recommended molecular assays for germ cell tumors include karyotyping of patients with symptoms of gonadal dysgenesis, gonadoblastoma and mediastinal tumors (for boys) and bilateral ovarian tumors (for girls), in order to detect/rule out the presence of genetic syndromes such as Turner, Swyer, Klinefelter, Fraser, Denys-Drash and others. The literature data also indicate the presence of somatic rearrangements involving chromosomes 1, 6, 11, 12, 16, 20, and 22, the clinical significance of which is currently unknown.
The standards were developed following the guidelines of the French TGM-95 protocol (1995), protocols elaborated by the international expert consortium MaGIC-Malignant Germ Cell International Collaborative and literature data [62].

Liver Tumors
The most common pediatric liver tumor is hepatoblastoma. The incidence of hepatoblastoma is 2-3 cases per 1 million children under 15 years of age; however, approximately The diagnostic standards for hepatoblastoma according to the International Childhood Liver Tumors Strategy Group (SIOPEL) were included in forming these guidelines. A set of clinically relevant molecular markers for hepatic tumors of childhood age is provided in Table 6 [64,70,[78][79][80]. 98% of all cases occur in children under 5 years of age. The mean age at diagnosis is 18 months [63]. Survival rates for children with hepatoblastoma exceed 80%. Molecular alterations reported in hepatoblastoma primarily involve genes, the protein products of which regulate the WNT and NF-κB pathway. Thus, the most common somatic alterations are those in genes: CTNNB1 (80-90%), APC (2-3%), AXIN1, AXIN2 and PIK3CA as well as TERT (2-6%) and NFE2L2 (5-10%) [63][64][65][66][67][68][69]. The results of chromosomal rearrangement analysis indicate that this group of cancers has a higher rate of somatic rearrangements within chromosomes 1, 2, 8, and 20. However, the clinical significance of their occurrence is still unknown [64,65,70]. One of the objectives of the Pediatric Hepatic International Tumor Trial (PHITT (NCT03017326)), which has been running since 2017, is to determine the clinical significance of molecular findings including complete genomic, transcriptomic, and epigenomic profiling for hepatoblastoma patients [71].
Other hepatic tumors of childhood include: hepatocellular carcinoma (HCC) which is very rarely diagnosed in children, usually in older patients (10-14 years), but has been also found in children younger than 5 years of age. The prognosis is dismal, after conservative surgical treatment (30% of children achieving 3-year survival). Liver transplantation in children with HCC contributed to the improvement of overall survival (>70%). The most frequently reported somatic alterations in this cancer are alterations in the TERT and TP53 genes (60% and 25-30%, respectively); however, the molecular background is still unknown [72,73]; • undifferentiated embryonal sarcoma of the liver (UES) which is a rare liver tumor with onset in children mostly aged between 6 and 10 years. The molecular background of this neoplasm is not fully understood; however, according to literature data, the characteristic features of this neoplasm comprise frequent and extensive chromosome rearrangements, also in the form of chromothripsis [74]. Additionally, alterations were observed within the 19q13.4 region, including a t(11;19)(q13;q13.4) translocation and overexpression of the C19MC region (miRNA cluster). The presence of TP53 gene alterations was also observed [75][76][77][78].
The diagnostic standards for hepatoblastoma according to the International Childhood Liver Tumors Strategy Group (SIOPEL) were included in forming these guidelines. A set of clinically relevant molecular markers for hepatic tumors of childhood age is provided in Table 6 [64,70,78-80].

Retinoblastoma
Retinoblastoma is the most common primary malignant intraocular cancer in children and the second most common cancer of the eye in all age groups after choroidal melanoma. It accounts for 3% of all childhood tumors. The number of cases ranges from 1 in 14,000-1 in 18,000 live births [3,81]. The following forms of retinoblastoma are distin-

Retinoblastoma
Retinoblastoma is the most common primary malignant intraocular cancer in children and the second most common cancer of the eye in all age groups after choroidal melanoma. It accounts for 3% of all childhood tumors. The number of cases ranges from 1 in 14,000-1 in 18,000 live births [3,81]. The following forms of retinoblastoma are distinguished as: Most cases are diagnosed between the ages of 1 and 3 years. The bilateral form of retinoblastoma is diagnosed earlier, before the age of 1. Rarely, the disease is diagnosed after the age of 5 years.
This cancer is associated with high (85-95%) penetrance RB1 gene alterations [81]. There are recent reports of molecular alterations in retinoblastoma patients in genes other than RB1-   Most cases are diagnosed between the ages of 1 and 3 years. The bilateral form of retinoblastoma is diagnosed earlier, before the age of 1. Rarely, the disease is diagnosed after the age of 5 years.
This cancer is associated with high (85-95%) penetrance RB1 gene alterations [81]. There are recent reports of molecular alterations in retinoblastoma patients in genes other than RB1-

Prognostic, Predictive Markers
Retinoblastoma Melanoma is a malignant neoplasm of the skin, mucous membranes, or the choroid of the eye originating from melanocytes. The incidence is 1 case per 1 million children under 15 years of age. In the pediatric group, melanoma can present with one of three types: • Spitzoid melanoma (SM), the most common form, • Melanoma that arises from a congenital melanocytic nevus (CMN) • Classic melanoma ("adult-type melanoma"), most similar in terms of causes and risk factors to melanoma diagnosed in adults.
Most commonly, melanoma is associated with molecular changes in genes that regulate the MAPK pathway. The recommendations were prepared following the current literature data. A set of molecular markers clinically relevant for childhood melanoma is provided in Table 8 [86][87][88][89][90][91].

Melanoma
Melanoma is a malignant neoplasm of the skin, mucous membranes, or the choroid of the eye originating from melanocytes. The incidence is 1 case per 1 million children under 15 years of age. In the pediatric group, melanoma can present with one of three types: • Spitzoid melanoma (SM), the most common form, • Melanoma that arises from a congenital melanocytic nevus (CMN) • Classic melanoma ("adult-type melanoma"), most similar in terms of causes and risk factors to melanoma diagnosed in adults.
Most commonly, melanoma is associated with molecular changes in genes that regulate the MAPK pathway. The recommendations were prepared following the current literature data. A set of molecular markers clinically relevant for childhood melanoma is provided in Table 8 [86][87][88][89][90][91].

Ovarian Cancers
The occurrence of ovarian cancer in girls may be associated with syndromes such as DICER1 or RTPS (rhabdoid tumor predisposition syndrome); therefore, both germ cell and somatic alterations are also observed in DICER1 (sertoli-Leydig cell tumors) and SMARCA4 (primary small cell carcinoma of the ovary, hypercalcemic type-SCCOHT) ( Table 9) [92]. TERT (promoter changes) + segmental rearrangements within chromosomes -

Ovarian Cancers
The occurrence of ovarian cancer in girls may be associated with syndromes such as DICER1 or RTPS (rhabdoid tumor predisposition syndrome); therefore, both germ cell and somatic alterations are also observed in DICER1 (sertoli-Leydig cell tumors) and SMARCA4 (primary small cell carcinoma of the ovary, hypercalcemic type-SCCOHT) ( Table 9) [92].

Targeted Treatments for Pediatric Solid Tumours
Treatments for pediatric malignancies have changed vastly over the last several decades and cure rates now reach over 80%. However, there are still children with uncurable malignancies and those who are cured experience treatment related chronic health conditions. The progress in the field of molecular biology, the ability to analyze tissue on genome-wide scales, to identify cancers with specific gene alterations with the intent to develop novel targeted treatments has created new opportunities to further improve survival of childhood cancer patients and their quality of life. To date the use of targeted and immune-therapies in children has been limited. Despite many obstacles of drug development in pediatric oncology some medicinal products have come to the market and are used in front-line treatment. There are ongoing pediatric phase I/II biomarker-driven trials in most difficult to treat solid tumors in children. Table 10 presents selected targeted treatments authorized or in development.

Targeted Treatments for Pediatric Solid Tumours
Treatments for pediatric malignancies have changed vastly over the last several decades and cure rates now reach over 80%. However, there are still children with uncurable malignancies and those who are cured experience treatment related chronic health conditions. The progress in the field of molecular biology, the ability to analyze tissue on genome-wide scales, to identify cancers with specific gene alterations with the intent to develop novel targeted treatments has created new opportunities to further improve survival of childhood cancer patients and their quality of life. To date the use of targeted and immune-therapies in children has been limited. Despite many obstacles of drug development in pediatric oncology some medicinal products have come to the market and are used in front-line treatment. There are ongoing pediatric phase I/II biomarker-driven trials in most difficult to treat solid tumors in children. Table 10 presents selected targeted treatments authorized or in development.

Germline Alterations
In recent years, we have been witnessing great progress in understanding the molecular profile of childhood cancers and applying this knowledge to clinical practice. This includes somatic as well as germline alterations. The published findings of different multiomics studies further highlight the differences between childhood and adult cancers. The global number of somatic alterations, as expressed by the Tumor Mutational Burden (TMB), in childhood malignancies is much lower than in adults. With respect to germline alterations, the opposite is true. The occurrence of childhood cancers is more often determined by the presence of alterations responsible for genetic syndromes. More than 200 such syndromes have been identified, and this number is steadily growing. The fact that congenital cancers are also diagnosed underscores the significance of germline alterations in the pathogenesis of childhood cancers. It is estimated that approximately 7-8% of hematologic malignancies and solid tumors in children are determined by germline alterations [93]. Furthermore, these data appear to be underestimated since germline mosaicism, which is difficult to identify, or epigenetic changes, such as loss of imprinting of the 11p15 region or hypermethylation of the CDKN2A suppressor, are rarely assessed in the routine diagnosis of cancer.
Assessment of the presence of germline alterations is also important in optimizing therapeutic management. If patients present with lesions that result from chromosomal instability or that occur in DNA repair genes, the omittance of radiotherapy or a reduced radiation dose are recommended. One such example is children with choroid plexus carcinoma and germline alterations in TP53 gene. Patients with germline RB1 alterations who underwent radiotherapy have twice the risk of developing secondary cancers compared with patients who did not receive radiotherapy [94]. The presence of germline alterations in the NF1 gene in patients with low-grade gliomas is associated with a better prognosis; hence, the treatment undertaken may be less aggressive [95]. However, patients with germline alterations in genes belonging to the (MMR mismatch repair system) such as MLH1, MSH2, MSH6, and PMS2 with brain cancers require more aggressive chemotherapy [96]. The presence of a germline alterations may also contribute to the earlier cancer onset. Rhabdoid tumors occur more frequently in patients under 4 years of age; however, the mean age of onset in patients with germline SMARCB1 alterations is 6 months [97,98]. Therefore, it seems highly appropriate to introduce the assessment of germinal alteration status into the algorithm of diagnostic and therapeutic management of pediatric cancers.
The following tables (Tables 11 and 12) present selected genetic syndromes associated with the occurrence of childhood cancers, as well as information about which genes should be assessed in selected cancers of this age group.

Material and Conditions for Its Preservation for Genetic Testing
The starting point for most oncogenetic tests used is a tumor tissue sample. Formalin fixation and paraffin embedding (FFPE) is the most common form of tissue preservation; however, for NGS-based protocols, tissue preservation by freezing is much more beneficial. In selected tumors (e.g., neuroblastoma), the impression smear of tumor tissue may serve as the material for genetic assay. To identify germline alterations, the patient's peripheral blood, saliva and/or buccal swab samples are also collected in addition to the tumor tissue.
Due to the heterogeneous nature of tumors (particularly neuroblastoma and selected gliomas), it is recommended that at least two specimens be collected from the respective tumor tissue. In cases of tumor recurrence and probable changes in the molecular profile of the relapsed tumor tissue, resampling is indicated.
Each tumor tissue specimen for molecular assay should be evaluated for the percentage of tumor cells in the tested specimen. This assessment is routinely performed by a pathologist. If more than one biological sample is available, the most appropriate sample should be selected based on the type of molecular assay planned, the availability of the biological material and the need for it at subsequent stages of diagnostic process.
It is also very important to maintain sterility when collecting material for molecular assays. NGS methods can detect mosaic-type alterations at very low levels. In cases of contamination of the specimen with even a very small amount of material from another patient, false results may be obtained.

Methylation Profile
The rapid development of high-throughput next-generation sequencing methods has significantly contributed to the understanding of the molecular profile of the most common pediatric cancers. Today, we know that, on the one hand, these tumors exhibit a great variety of molecular alterations, but, on the other hand, their total number is small compared to the number of somatic alterations detected in tumors occurring in adults. This is reflected very frequently in the low TMB score, which translates into limited applicability of immunotherapy. Effective immunotherapy can also be limited by the suppressive tumor microenvironment with relatively few effector cells. Generally, this type of therapy in pediatric solid tumors still remains in the early stages of development and significant clinical benefit has yet to be demonstrated.
In contrast, epigenetic changes occupy a special place in the vast spectrum of molecular alterations that are identified in childhood cancers. It seems that this type of alteration is crucial for the initiation of carcinogenesis-related processes. A confirmation of this assumption is the occurrence of different genome DNA methylation patterns in different tumor subtypes. A unique epigenetic signature that represents both the tumor origin and the presence of acquired oncogenic alterations affecting chromatin state constitutes a very promising diagnostic tool for, among others, central nervous system tumors in children. The classification system developed by German National Cancer Institute (DKFZ) in Heidelberg, based on the methylome pattern [99] is slowly becoming a routine tool to accurately classify CNS tumors into distinct molecular subtypes. As a result, it may improve the accuracy of diagnosis and standardize pathomorphological assessment. This diagnostic approach is limited by the requirement for specialized equipment, software, and a large reference database. To address this problem, the DKFZ team developed a free online tool, Classifier [100], which allows for the processing of data obtained in a given laboratory and comparing them to a reference database containing the results of methylation profile analysis from over 2800 cases. An additional advantage of this solution is the ability to verify the histopathological diagnosis in morphologically ambiguous cases and, in the future, also the ability to identify new, very rare tumor subtypes, not only of the central nervous system.

Liquid Biopsy
There is a growing number of published studies demonstrating the important role of liquid biopsy in the diagnostic and therapeutic management of a number of cancers, including pediatric malignancies. It is a complementary or alternative method to surgical biopsy, as well as a non-invasive, promising tool for early cancer detection, that may also overcome problems of tumor accessibility and heterogeneity of tumor tissue. Various biological fluids, including peripheral blood, urine, cerebrospinal, synovial and ocular space fluids, can be used to obtain such circulating material as tumor cells (CTCs), tumor DNA (cfDNA), RNA (cfRNA), proteins and extracellular vesicles (EVs) for diagnostic assays. Thanks to the advances in technology, it is possible to obtain and analyze such biological material with increased effectiveness. There are data demonstrating the usefulness of liquid biopsy in variety of analysis including small-and large-scale mutation analysis, high throughput sequencing technologies, and analysis of structural or copy number alterations. From a clinical perspective, the results from liquid biopsy can provide reliable data as to the status of the disease and allow us to monitor treatment and to evaluate predictive, prognostic and resistance markers. In some cases, it may be helpful in early detection of recurrence. Thus, liquid biopsy, although still a relatively new method, appears to be a significant application for cancer diagnosis and treatment. The results of published research on neuroblastoma, sarcoma, Wilms tumor, hepatoblastoma and retinoblastoma appear to be very promising [101]. Nevertheless, the implementation of liquid biopsy into clinical practice is still to be completed. Its limitations are mainly due to the lack of standardized, validated methods for such analyses and the rarity and instability of obtained tumor biomolecules. However, researchers agree that liquid biopsy represents a potentially major new method that can be used to detect, monitor and treat cancers. Further studies are required to address the limitations of this technique.

Conclusions
The introduction of technologies such as massively parallel DNA sequencing and RNA sequencing, as well as tools for the interpretation of the vast amounts of data obtained with these methods, including bioinformatic or crystallographic methods, creates an opportunity to elucidate the molecular mechanisms of childhood cancers and to develop targeted therapies. Artificial intelligence methods are also becoming increasingly employed to design therapeutic algorithms and identify prognostic and predictive markers [102]. The introduction of monitoring of circulating tumor DNA (ctDNA) using next-generation sequencing will enable future precise monitoring of treatment. The integration of a broad spectrum of data from "-omics" studies provides the basis for the development of cancerspecific classifiers used for precise diagnostics. More novel in vivo and in vitro models as well as 3D cultures are being developed and used to test drugs specifically dedicated to pediatric cancers. The aim of all of these efforts is to identify molecular markers and move them into the clinical setting for more precise diagnosis, risk stratification, and more effective and less toxic treatment in this therapeutically challenging group of patients.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare that there are no conflict of interest.